Vanadium compounds as anti-angiogenic agents

ABSTRACT

Vanadium compounds for inhibiting angiogenesis useful for treating or preventing diabetic retinopathy, hemangiomas, cancers with abnormal blood vessel supply, restenosis following vascular injury, and the like.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. patent applicationSer. No. 09/713,780, filed on Nov. 15, 2000, which application is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Tissue growth is intimately associated with blood supply.Non-vascularized tissue is limited in size, often being smaller than oneto two millimeters in diameter or thickness. Therefore, inhibiting bloodsupply to tissue represents one target point for limiting tissue growthand possibly tissue viability.

The ability to inhibit blood supply has been shown to play a pivotalrole in the progression, invasive and metastatic growth of malignanttumors (Folman, Nat Med, 1:27, 1995; Folkman et al, Science, 235:442,1985; Gimbrane et al, J Exp Med, 136:261, 1972). Further, inhibition ofangiogenesis new vessel formation), has been shown to result in tumordormancy or regression and to prevent metastasis (Folkman, Ann Surg,175:409, 1972; Taylar et al, Nature, 297:307, 1982).

Another possible target for inhibiting tissue growth is by inhibitingcell proliferation. All proliferating eucaryotic cells must undergomitosis before separating into two new cells. Mitosis is a process inwhich the parent or replicating cell undergoes a series of molecularevents that results in the formation of two nuclei in the place of one.

The polar mitotic spindle is critical to the separation of thereplicated chromosomes and formation of the two nuclei in the mitoticprocess. For mitosis to proceed normally, cells must properly form abipolar mitotic spindle with bivalent chromosomes properly attached toeach pole of the spindle (Gorbsky et al, Bioessays, 19: 193-197, 1997;Hardwick, K. G., Trends Genet., 14: 1-4, 1998). Cells which do not forma correct mitotic spindle arrest indefinitely in the metaphase stage ofmitosis or progress into apoptosis. Several proteins identified in yeastand mammals have been implicated in this process, including MAD1(mitotic arrest deficient), MAD2, and MAD3 (Li et al, Cell, 66: 519-531,1991 (published erratum appears in Cell, 79(2), following p388)), BUB1(budding uninhibited by benzimidazole), BUB2 and BUB3 (Hoyt et al, Cell,66: 507-517, 1991). Mammalian counterparts for these proteins includeHsMAD2 (Li et al, Supra and hBUB1 (Cahill et al, Nature, 392: 300-303,1998).

Revascularization of obstructed coronary arteries by percutaneoustransluminal coronary angioplasty (PTCA) has become an integralcomponent of front-line treatment programs for patients with ischemicheart disease (Vaitkus, P. T., 1995, Coron. Atery Dis., 6:429-439).Although acute complications of PTCA have markedly declined withoptimized use of anticoagulants, antispasmodic agents, and intravascularstents, the incidence of coronary artery restenosis has remained at30%-50% and represents the major obstacle to a more successful outcomeof PTCA (Landzberg, et. al., 1997, Prog. Cardiovascular Diseases,39:361-298). Therefore, the development of effective strategies forrestenosis prophylaxis has become a focal point for translationalcardiovascular research.

The pathogenesis of restenosis has been compared to an exaggerated woundhealing response with migration of smooth muscle cells from the media tothe intima of the revascularized coronary artery where they proliferateand cause an obstructive neointimal hyperplasia (Ueda et al., 1995,Coron. Artery Dis., 6:71-81). Many factors contribute to the developmentof restenosis, including vascular injury, platelet aggregation,procedural factors, inflammation, and mitogenic stimulation of migrationand proliferation of smooth muscle cells. The relative contribution ofany one of these factors remains unclear.

Pharmacological approaches to prevent restenosis include antiplateletand antithrombotic agents, anti-inflammatory drugs, growth factorantagonists, vasodilators, antiproliferatives, antineoplastics,photochemotherapy, and lipid lowering agents. Some growth factorantagonists have also been studied for effects on restenosis.

Inhibition of vascular smooth muscle cell proliferation by a plateletderived growth factor (PDGF)-antagonist has generated promising resultsin preclinical as well as early clinical studies, thereby confirming thebiologic importance of vascular smooth muscle cells in thepathophysiology of restenosis (Mullins et al., 1994, Arterioscler.Thromb., 14:1047-1055).

Considerable efforts are underway to develop new anti-angiogenic andanti-mitotic agents for use as therapies in the treatment of tumorgrowth and spread. Accordingly, there is a need for the analysis ofnovel, effective anti-angiogenic and anti-mitotic agents that targettumor growth.

Vanadocene dichloride (VDC) has been shown to arrest tumor cells growth(Kopf-Maier, et al, J Cancer Res. Clin. Onccol., 106: 44-52, 1983). Theoxovanadium compound, VO(Phen)(H₂O)2](SO₄), has been shown to be anactive agent against pharyngonasal cancer as determined by a singleassay (Sakurai, et. al, BBRC, 206: 133, 1995). Vanadium compounds,including vanadocenes, have also been demonstrated to induce apoptosisin cancer cells (Uckun et al., WO 00/35930).

Against this backdrop the present invention has been developed.

SUMMARY OF THE INVENTION

It has been now found that vanadium compounds, for example vanadocenesand oxovanadium compounds, are poent agents for inhibiting angiogenesis.In particular, the vanadium compounds have been found to exhibit dualfunction inhibiting both angiogenesis and mitosis. Pharmaceuticalcompositions containing these vanadium compounds are thus useful inmethods to inhibit angiogenesis and mitosis, for example, in thetreatment diabetic retinopathy, hemangiomas, cancers with abnormal bloodvessel supply, restenosis following vascular injury, and the like.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention relates to vanadium compounds, including, forexample, vanadium cyclopentadienyl complexes (vanadocenes), such asvandocene acetylacetonate (VDacac) and oxovanadium compounds, and thefinding that these vanadium compounds have potent anti-angiogenic andanti-mitotic activities.

Vanadium is a physiologically essential element that can be found inboth anionic and cationic forms with oxidation states ranging from −3 to+5 (I-V). This versatility provides unique properties to vanadiumcomplexes. In particular, the catonic form of vanadium complexes havingan oxidation state of +4 (IV) has been shown to function as a modulatorof cellular redox potential, regulate enzymatic phosphorylation, andexert pleiotropic effects in multiple biological systems by catalyzingthe generation of reactive oxygen species (ROS). Besides the ability ofvanadium metal to assume various oxidation states, its coordinationchemistry also plays a key role in its interactions with variousbiomolecules. In particular, it is demonstrated herein that vanadiumcompounds, such as vanadium cyclopentadienyl complexes, oxovanadiumcomplexes, or derivatives thereof, exhibit anti-angiogenic andanti-mitotic properties.

Definitions

The following terms and phrases as used herein have the noteddefinitions, unless otherwise described:

“Halo” is fluoro, chloro, bromo, or iodo. “Alkyl”, “alkoxy”, etc. denoteboth straight and branched hydrocarbon groups; but reference to anindividual radical such as “propyl” embraces only the straight chainradical, a branched chain isomer such as “isopropyl” is specificallyreferenced.

“Organometallic compound” is an organic compound comprised of a metalattached directly to carbon (R-M).

“Coordination compound” is a compound formed by the union of a centralmetal atom or ion with ions or molecules called ligands or complexingagents.

“Ligand” or a “complexing agent” is a molecule, ion or atom that isattached to the central metal atom or ion of a coordination compound.

“Monodentate ligand” is a ligand having a single donor atom coordinatedto the central metal atom or ion.

“Bidentate ligand” is a ligand having two donor atoms coordinated to thesame central metal atom or ion.

“Vanadocene” is a compound having a central vanadium metal ioncoordinated with at least two cyclopentadiene groups.

It will be appreciated that compounds of the invention having a chiralcenter may exist in and be isolated in optically active and racemicforms. Some compounds may exhibit polymorphism. It is to be understoodthat the present invention encompasses any racemic, optically-active,polymorphic, or stereoisomeric form, or mixtures thereof, of a compoundof the invention, which possess the useful properties described herein.Methods to prepare optically active forms are known, for example, byresolution of the racemic form by recrystallization techniques, bysynthesis from optically-active starting materials, by chiral synthesis,or by chromatographic separation using a chiral stationary phase.Methods for determining anti-mitotic and anti-meiotic activity of acompound are known, for example, using the standard tests describedherein, or other known tests.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

For example, (C₁-C₆) alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl; (C₁-C₃) alkylcan be methyl, ethyl or propyl; halo(C₁-C₃) alkyl can be iodomethyl,bromomethyl, chloromethyl, fluoromethyl, trifluoromethyl, 2-chloroethyl,2-fluoroethyl, 2,2,2-trifluoroethyl, or pentafluoroethyl; (C₁-C₃) alkoxycan be methoxy, ethoxy, or propoxy; and (C₂-C₆)alkanoyloxy can beacetoxy, propanoyloxy, butanoyloxy, isobutanoyloxy, pentanoyloxy, orhexanoyloxy.

The following glossary of vanadium compounds is provided to clarifyterms used throughout the specification and provides a listing ofexemplary vanadium compounds useful in the method invention:

Group A: Vanadocene Dihalides VDC Vanadocene dichloride (Cp₂VCl₂) VMDCBis (methyl cyclopentadienyl) vanadium dichloride [(MeCp)₂VCl₂] VDBVanadocene dibromide (Cp₂VBr₂) VDI Vanadocene diiodide (Cp₂VI₂)

Group B: Vanadocene Di-pseudohalides VDA Vanadocene diazide [Cp₂V(N₃)₂]VDCN Vanadocene dicyanide (Cp₂V(CN)₂) VDOCN Vanadocene dioxycyanate(Cp₂V(OCN)₂) VDSCN Vanadocene dithiocyanate (Cp₂V(SCN)₂) VDSeCNVanadocene diselenocyanate (VCp₂(SeCN)₂)

Group C: Vanadocene Disubstituted Derivatives VDT Vanadocene ditriflate(Cp₂V(O₃SCF₃)₂) VDCO Vanadocene monochloro oxycyanate (Cp₂V(OCN)(Cl))VDFe Vanadocene monoacetonitrilo monochloro tetrachloro ferrate(Cp₂VClNCCH₃)FeCl₄

Group D: Chelated Vanadocene Complexes VDacac Vanadocene acetylacetonatomonotriflate (Cp₂V(CH₃COCH₂COCH₃)(O₃SCF₃) VDBPY Vanadocene bipyridinoditriflate (CP₂V(C₁₀H₈N₂)(O₃SCF₃)₂) VDHfacac Vanadocene hexafluoroacetylacetonato monotriflate Cp₂V(CF₃COCH₂COCF₃)(O₃SCF₃)) VDH Vanadoceneacethydroxamato monotriflate (Cp₂V(CH₃CON(O)H)(O₃SCF₃) VDPH VanadoceneN-phenyl benzohydroxamato monotriflate (Cp₂V(C₆H₅CON(O)C₆H₅)(O₃SCF₃)

Group E. Oxovanadium Compounds

[(VO(phen)]=(diaqua)(1,10-phenanthroline)oxovanadium (IV) sulfate;

[VO(phen)₂]=(aqua)bis(1,10-phenanthroline)oxovanadium (IV) sulfate;

[VO(Me₂-phen)]=(diaqua)(4,7-dimethyl-1,10-phenanthroline)oxovanadium(IV) sulfate;

[VO(Me₂-phen)₂]=(aqua)bis(4,7-dimethyl-1,1 0-phenanthroline)oxovanadium(IV) sulfate;

[VO(Cl-phen)]=(diaqua)(5-chloro-1,10-phenanthroline)oxovanadium (IV)sulfate;

[VO(Cl-phen)₂]=(aqua)bis(5-chloro-1,10-phenanthroline)oxovanadium (IV)sulfate;

[VO(bipy)]=(diaqua)(2,2′-bipyridyl)oxovanadium (IV) sulfate;

[VO(bipy)₂]=(aqua)bis(2,2′-bipyridyl)oxovanadium (IV) sulfate;

[VO(Me₂-bipy)]=(diaqua)(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium(IV)sulfate;

[VO(Me₂-bipy)₂]=(aqua)bis(4,4′-dimethyl-2,2′-bipyridyl)oxovanadium (IV)sulfate;

[VO(Br,OH-acph)₂]=bis(5′-bromo-2′-hydroxyacetophenone)oxovanadium (IV).

Unless otherwise indicated, the following abbreviations are usedthroughout the remainder of the disclosure:

Cp, cyclopentadienyl

Cp⁻, cyclopentadienyl anion

acac, acetonylacetonate

Bpy, 2,2′ Bipyridine

Hfacac, hexafluoroacetylacetonate

Cat, catecholate

Dtc, diethyl dithio carbamate

Phen, penanthroline

PH, N-phenyl benzohydroxamic acids

H, acethydroxamic acid

OTf, trifluoromethane sulphonate

THF, tetrahydrofuran

DMSO, dimethyl sulfoxide

CH₃CN, acetonitrile

CH₂Cl₂, dichloromethane

d-d, laportte spin forbidden transitions

LMCT, ligand to metal charge transfer transitions

p-p*, intraligand charge transfer transitions

The present invention concerns vanadium compounds, and the finding thatsuch compounds have potent and selective anti-mitotic activity, and areparticularly active and stable agents for inhibiting angiogenesis. Assuch, these compounds are useful to treat or prevent disorders such asdiabetic retinopathy, hemangiomas,cancers with abnormal blood vesselsupply, restenosis following vascular injury, and the like.

Vanadium (IV) compounds for use in this invention are as shown informula I and formula II:

where R¹ and R₂ are each independently a monodentate ligand or togetherform a bidentate ligand; R₃ and R₄ are each independently a monodentateligand or together form a bidentate ligand; and R₅ is a monodentateligand, or is absent.

Suitable monodentate ligands include monodentate ligands are selectedfrom the group consisting of halo, OH₂, O₃SCF₃, N₃, CN, OCN, SCN, SeCN,and a cyclopentadienyl ring, wherein each cyclopentadienyl ring isoptionally substituted with one or more (C₁-C₃)alkyl. Suitable bidentateligands are selected from the group consisting of acac, Bpy, Hfacac,Cat, Dtc, PH, H, and Phen, or derivatives thereof. The bidentate ligandsmay be substituted, for example, with one or more (C₁-C₃) alkyl, halo,(C₁-C₃) alkoxy, and halo (C₁-C₃) alkyl, and derivatives thereof. Halo ischloro, bromo, or iodo, and preferably is chloro.

In one embodiment, a useful vanadium compound has the followingstructure:

where R₁ and R₂ are each independently a monodentate ligand or togetherform a bidentate ligand; and R₃ and R₄ are each independently acyclopentadienyl ring, wherein each cyclopentadienyl ring is optionallysubstituted with one or more (C₁-C₃)alkyl. In some preferredembodiments, R₁ and R₂ are each independently a monodentate ligandselected from the group consisting of of halo, OH₂, O₃SCF₃, N₃, CN, OCN,SCN, SeCN, and a cyclopentadienyl ring, where each cyclopentadienyl ringis optionally substituted with one or more (C₁-C₃)alkyl. Preferably, R₁and R₂ are halo, and more preferably chloro. In some other embodiments,R₁ and R₂ together form a bidentate ligand selected from the groupconsisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, and Phen, orderivatives thereof. Preferably, the bidentate ligand is acac, orderivatives thereof.

Some specific examples of compounds of formula I are: VCp₂Cl₂(VDC),VCp₂Br₂, VCp₂I₂, VCp₂(N₃)_(2,) VCp₂(CN)₂, VCp₂(NCO)₂, VCp₂(NCO)Cl,VCp₂(NCS)₂, VCp₂(NCSe)₂, VCp₂Cl (CH₃CN)(FeCl₄), VCp₂(O₃SCF₃)₂,V(MeCp)₂Cl₂, V(Me₅Cp)₂Cl₂, VCp₂(acac) (VDacac), VCp₂(hf-acac),VCp₂(bpy), VCp₂(cat), VCp₂(dtc), VCp₂PH, or VCp₂H. Two particularlyuseful vandocene compounds are VDC and VDacac.

Examples of useful oxovanadium compounds of formula II include thecompound having the following structure:

where R₁ and R₂ are each independently a monodentate ligand or togetherform a bidentate ligand; R₃ and R₄ together form a bidentate ligand; andR₅ is a monodentate ligand, or is absent. In some preferred embodiments,R₁ and R₂ are each independently a monodentate ligand selected from thegroup consisting of halo, OH₂, O₃SCF₃, N₃, CN, OCN, SCN, SeCN, and acyclopentadienyl ring, wherein each cyclopentadienyl ring is optionallysubstituted with one or more (C₁-C₃)alkyl, and R₃ and R₄ together form abidentate ligand selected from the group consisting of acac, Bpy,Hfacac, Cat, Dtc, PH, H, and Phen, or derivatives thereof. Inembodiments where R₁ and R₂ together form a bidentate ligand, thebidentate ligand is preferably selected from the group consisting ofacac, Bpy, Hfacac, Cat, Dtc, PH, H, and Phen, or a derrivative thereof.

Specific compounds of formula II include [VO(phen)], [VO(phen)₂],[VO(Me₂-phen)], [VO(Me₂-phen)₂], [VO(Cl-phen)], [VO(Cl-phen)₂ ],[VO(bipy)], [VO(bipy)₂], [VO(Me₂-bipy)], [VO(Me₂-bipy)₂], and[VO(Br,OH-acph)₂].

The vandocene compounds can be used as anti-angiogenesis andanti-mitosis agents. In some embodiments, such compounds are used in thetreatment of disorders in animals, and in particular in the treatment ofdiabetic retinopathy, hemangiomas, cancers with abnormal blood vesselsupply, restenosis following vascular injury, and the like.

In such cases, the compounds both inhibit angiogenesis, and act as ananti-mitotic agent within the cells. In this manner the compounds of thepresent invention are acting in a dual function, to both reduce theblood supply and to disrupt mitosis in dividing cells.

Administration of the compounds as salts may be appropriate. Examples ofacceptable salts include alkali metal (for example, sodium, potassium orlithium) or alkaline earth metal (for example calcium) salts, however,any salt that is non-toxic and effective when administered to the animalbeing treated is acceptable.

Acceptable salts may be obtained using standard procedures well known inthe art, for example by reacting a sufficiently acidic compound with asuitable base affording a physiologically acceptable anion.

The compositions of the invention can be formulated as pharmaceuticalcompositions and administered to an animal host, such as a human patientin a variety of forms adapted to the chosen route of administration,i.e., orally or parenterally, by intravenous, intramuscular, topical orsubcutaneous routes.

Thus, the present compounds may be systemically administered, e.g.,orally, in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, may be compressed intotablets, or may be incorporated directly with the food of thepatient'diet. For oral therapeutic administration, the active compoundmay be combined with one or more excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. Such compositions andpreparations should contain at least 0.1% of active compound. Thepercentage of the compositions and preparations may, of course, bevaried and may conveniently be between about 2 to about 60% of theweight of a given unit dosage form. The amount of active compound insuch therapeutically useful compositions is such that an effectivedosage level will be obtained. When administered orally, thecompositions of the invention can preferably be administered in agelatin capsule.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Of course, any material used in preparing anyunit dosage form should be pharmaceutically acceptable and substantiallynon-toxic in the amounts employed. In addition, the active compound maybe incorporated into sustained-release preparations and devices.

The compositions of the invention may also be administered intravenouslyor intraperitoneally by infusion or injection. Solutions of the activecomposition can be prepared in water, optionally mixed with a nontoxicsurfactant. Dispersions can also be prepared in glycerol, liquidpolyethylene glycols, triacetin, and mixtures thereof and in oils. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient which are adapted for theextemporaneous preparation of sterile injectable or infusible solutionsor dispersions, optionally encapsulated in liposomes. In all cases, theultimate dosage form should be sterile, fluid and stable under theconditions of manufacture and storage. The liquid carrier or vehicle canbe a solvent or liquid dispersion medium comprising, for example, water,ethanol, a polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycols, and the like), vegetable oils, nontoxic glycerylesters, and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the formation of liposomes, by themaintenance of the required particle size in the case of dispersions orby the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, buffers or sodiumchloride. Prolonged absorption of the injectable compositions can bebrought about by the use in the compositions of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecomposition in the required amount in the appropriate solvent withvarious of the other ingredients enumerated above, as required, followedby filter sterilization. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying techniques, whichyield a powder of the active ingredient plus any additional desiredingredient present in the previously sterile-filtered solutions.

For topical administration, the present compositions may be applied inpure form, i.e., when they are liquids. However, it will generally bedesirable to administer them to the skin as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid.

Useful solid carriers include finely divided solids such as talc, clay,microcrystalline cellulose, silica, alumina and the like. Useful liquidcarriers include water, alcohols or glycols or water-alcohol/glycolblends, in which the present compounds can be dissolved or dispersed ateffective levels, optionally with the aid of non-toxic surfactants.Adjuvants such as fragrances and additional antimicrobial agents can beadded to optimize the properties for a given use. The resultant liquidcompositions can be applied from absorbent pads, used to impregnatebandages and other dressings, or sprayed onto the affected area usingpump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Examples of useful dermatological compositions which can be used todeliver the compounds of formula I to the skin are known to the art; forexample, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat.No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman(U.S. Pat. No. 4,820,508).

Useful dosages of the compounds of the present invention can bedetermined by comparing their in vitro activity, and in vivo activity inanimal models. Methods for the extrapolation of effective dosages inmice, and other animals, to humans are known to the art; for example,see U.S. Pat. No. 4,938,949.

Generally, the concentration of the compositions of the invention in aliquid composition, such as a lotion, will be from about 0.1-50 wt-%,preferably from about 0.5-5 wt %. The concentration in a semi-solid orsolid composition such as a gel or a powder will be about 0.1-5 wt-%,preferably about 0.5-2.5 wt-%.

The amount of the composition required for use in treatment will varynot only with the particular salt selected but also with the route ofadministration, the nature of the condition being treated, and the ageand condition of the patient, and will ultimately be at the discretionof the attendant physician or clinician.

In general, however, a suitable dose will be in the range of from about0.1 to about 150 mg/kg, e.g., from about 10 to about 75 mg/kg of bodyweight per day, such as 3 to about 50 mg per kilogram body weight of therecipient per day, preferably in the range of 1 to 100 mg/kg/day, mostpreferably in the range of 5 to 20 mg/kg/day.

The compositions are conveniently administered in unit dosage form; forexample, containing 5 to 1000 mg, conveniently 10 to 750 mg, mostconveniently, 50 to 500 mg of active ingredient per unit dosage form.

Ideally, the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.5 to about75 μM, preferably, about 1 to 50 μM, most preferably, about 2 to about30 μM. This may be achieved, for example, by the intravenous injectionof a 0.05 to 5% solution of the active ingredient, optionally in saline,or orally administered as a bolus containing about 1-100 mg of theactive ingredient. Desirable blood levels may be maintained bycontinuous infusion to provide about 0.01-5.0 mg/kg/hr or byintermittent infusions containing about 0.4-15 mg/kg of the activeingredient(s).

The desired dose may conveniently be presented in a single dose or asdivided doses administered at appropriate intervals, for example, astwo, three, four or more sub-doses per day. The sub-dose itself may befurther divided, e.g., into a number of discrete loosely spacedadministrations; such as multiple inhalations from an insufflator or byapplication of a plurality of drops into the eye.

Following i.m. administration, the compositions of the invention enterthe blood stream within about 10-15 minutes and reach a maximumconcentration in the blood within one hour of administration, at whichpoint they can be found throughout the circulatory related organs.

Note, for the examples that follow, Tetraphenyl borate,trifluoromethanesulfonato and acetyl acetone were purchased from AldrichChemical Co. (Milwaukee, Wis.). Other reagents used were of commerciallyavailable reagent grade quality, and unless otherwise stated, allsolvents were used as received from Aldrich (Sure Seal bottle, <0.005%water). Tetrahydrofuran was dried by distillation over sodium. Infraredspectral data were recorded using a FT-Nicolet model Protege 460 andtaken as a KBr pellet. Frequencies were generally in the range of4000-500 cm⁻¹. UV-Vis spectra were recorded in a quartz cell on aBeckman Model DU 7400 spectrophotometer and the spectral bands wereregistered in the the 250-800 nm range. Electron paramagnetic resonance(EPR) spectra were recorded in standard PBS buffer on a Bruker ESP 300EPR spectrophotometer (9.64 GHz) using 4102 standard cavity. The gvalues were calibrated with a Varian strong pitch (0.1% in KCl) standard(g value 2.0028). The samples (in PBS) for EPR spectral analysis werestudied in Willmad WG-814 standard TE₁₀₂ aqueous cell cavity (0.3 mminner path length) to minimize the dielectric loss. Hyde, Rev SciInstrum, 43: 629, 1972. Magnetic moments were determined by Evans methodin CDCl₃ on a Varian 300 MHz FTNMR spectrometer. (Evans, J Chem Soc,2003, 1959).

In some embodiments, the compounds of the invention can be administeredbefore, during and/or after a vascular injury. Vascular injuries occurin human patients, for example, after medical procedures such ascoronary angioplasty. These percutaneous procedures are conducted onpatients with stable angina with single vessel disease, as well as thosewith multivessel disease, total occlusion, complex lesions, unstableangina and acute myocardial infarction. Several procedures in common usetoday can result in vessel injury, and would benefit from the method ofthe invention. These include balloon angioplasty, vessel stents,rotational and directional atherectomy, and laser angioplasty.

In a preferred embodiment, a patient is pretreated a vanadium compoundsuch as VDacac at least one to three days before the treatment orprocedure which is known to induce vascular injury. For example, it ispreferred that administration of the vanadium compound is performed oneto three days before a medical procedure such as PTCA. Delivery of thevanadium compound preferably continues after the vascular injury ormedical procedure up to a period of about 2 weeks to 6 months postinjury. The compound is preferably administered to the patient for aperiod of about 1 month to 3 months post injury. It is believed that theperiod 1 month to 3 months is the time at which restenosis formationpeaks after a vascular injury.

However, often the pretreatment option is not available to patients inan emergency situation. An emergency situation may arise requiring amedical procedure that causes vascular injury. In that situation, thevanadium compound can be administered during the procedure and/or afterthe procedure. The compound can be administered for a period afterinjury of 2 weeks to about 6 months, preferably at least 1 to 3 months.

The administration of the vanadium compound ameliorates or preventsdevelopment of restenosis. Some level of neointima hyperplasia can stillbe present in those patients treated with vanadium compounds, but theformation of neointima is significantly ameliorated compared tountreated patients. Treatment course and dose of the vanadium compoundscan be adjusted in the patient if the clinical signs indicate a need forincreased dose or treatment time.

Methods for analysis of restenosis are known, and include, for example,quantitative coronary angiography (QCA), where narrowing of a vessel isvisualized by injection of a visualizing dye. Another method foranalyzing restenosis is by intravascular ultrasound imaging (IVUS). Byinserting the ultrasound probe into a vessel, the diameter of thevessel, as well as the type and extent of lesions in the vessel can beanalyzed.

EXAMPLES

The present invention may be better understood with reference to theaccompanying examples that are intended for purposes of illustrationonly and should not be construed to limit the scope of the invention, asdefined by the claims appended hereto.

Example 1 Synthesis of Vanadium Compounds

Vanadium compounds useful in the invention may be prepared by knownmethods, as described, for example, in published PCT ApplicationsWO99/36063; WO 00/27389; and WO 00/35930. For example, VDC (VCp₂Cl₂)and. [VCp₂(acac)](CF₃SO₃); (VDacac) were prepared by followingliterature procedures (Wilkinson et al., J. Am. Chem. Soc., 76:4281-4284, 1954; Doyle et al., Inorg. Chem., 7: 2479-2484, 1968) andpurity was confirmed by ¹H NMR, IR spectroscopy, and elemental analysis.

Example 2 Inhibition of Angiogenesis

The present example illustrates that vanadium compounds such as[VCp₂(acac)](CF₃SO₃) are effective inhibitors of angiogenesis.

Chick Embryo Chorioallantoic (CAM) Assay

Inhibition of embryonic angiogenesis was determined using a bioassaysystem involving CAMs of growing chick embryos, as previously described(Nguyen et al, Microvascular Research, 47:31, 1994; Auerbach et al,Developmental Biology, 41:391, 1974). Fertilized white Leghorn chickeneggs were received at day 3 of incubation from the University ofMinnesota Poultry Research Center, St. Paul, Minn. The followingprocedures took place in a sterile laminar flow hood. The eggs werewiped down with 70% isoprpyl alcohol and allowed to air dry. The eggswere wiped with Betadine and placed in a horizontal position forapproximately 5 minutes and allowed to dry. The eggs were cracked andplaced into sterile 100×20 mm2 Petri dishes (Fischer, Itasca, Ill.) andtransferred to a 37° C. humidified incubator (1.5% CO₂). Pelletscontaining 1-100 μg [VCp₂(acac)](CF₃SO₃) in DMSO and 0.5%methylcellulose (Sigma, St. Louis, Mo.) were prepared by pipetting 10 μlof the compound, using a positive displacement pipette, onto sterileTeflon™PFA Petri dishes (VWR, Chicago, Ill.) and drying in a vacuumdesiccator at ambient temperature. The pellets containing the indicateddose of [VCp₂(acac)](CF₃SO₃) were implanted on the outer third of a 4-6day old CAM surface, generally between two branches of a prominent bloodvessel. The eggs were returned to a humidified 1.5% CO₂/37° C.incubator.

Inhibition of angiogenesis was assessed 48 hours after implantation bymeasuring the avascular zone in the CAM beneath the pellet, followed byphotographic documentation of the CAM. Pellets containing 200 μg suramin(Calbiochem, La Jolla, Calif.) served as a positive control. Themethylcellulose disk alone with no added compounds served as a negativecontrol. Significant inhibition of angiogenesis was defined as thepresence of an avascular zone of at least 3 mm in diameter around themethylcellulose disk.

Results The effect of [VCp₂(acac)](CF₃SO₃) on angiogenesis was examinedin standard chorioallantoic membrane (CAM) assays using 4-6 day oldchick embryos. [VCp₂(acac)](CF₃SO₃) in methylcellulose disks at dosesranging from 1 μg to 100 μg rapidly inhibited angiogenesis and producedwithin one hour a large avascular zone in the CAM around themethylcellulose disk, whereas control embryos implanted with empty discsdid not develop avascular zones. The average size of the avascular zoneincreased with increasing dose of the vanadium compound.

The data indicates that the vanadium compound [VCp₂(acac)](CF₃SO₃) is apotent inhibitor of angiogenesis. VDacac Avascular Zone (mg) (mm²)  17.0 ± 1.5  10 38.4 ± 16.6 100 92.4 ± 24.9

Example 3 Inhibition of Mitosis

This example illustrates that the vanadium compound [VCp₂(acac)](CF₃SO₃)is an effective inhibitor of embryonic development of Zebra fish, and inparticular, is a potent inhibitor of mitosis.

Zebra Fish and Embryos

The adult wild type ZF were maintained generally according to the“Zebrafish Book” recommendations (Westerfield, The Zebrafish Book, 1993,2d Edition Univ. of Oregon Press, Eugene). Males and females were keptin 10 Gallon tanks (70 fish per tank) with a constant slow flow ofconditioned water at 26° C. and a controlled 14 hour day/10 hour nightcycle. Adult fish were fed twice a day with live brine shrimp (OceanStar International, Snowville, Utah) and each group of fish was bredonce in two weeks. The embryos were obtained through (a) naturalspawning at 28.5° C. in the breeding tanks with a netted false bottom or(b) fertilization in vitro using eggs and milt collected from the maturefemales and males anesthetized with Tricaine (Sigma, St. Louis, Mo.), asdescribed (Westerfield, The Zebrafish Book, Supra).

Zebrafish Embryo Model System

ZF eggs were removed from their chorions by mild digestion in 1 %Trypsin-EDTA (Sigma, St. Louis, Mo.) for 10 minutes at 28.5° C.(Standard temperature—ST), washed three times in “egg water” and twicein “embryonic medium” (EM), according to recommendations (Westerfield,The Zebrafish Book, Supra). The dechorionated two-cell stage cleavingeggs were transferred to the 24-well plastic cell culture plates(Costar, Cambridge, Mass.) filled with EM. Dechorionated embryos (10-12per well) were exposed to the drugs at a constant ST for 0.5-24 hours.The final volume of the media in each well was 500 μL. The compound wastested at concentrations ranging from 10 μM to 4 mM. First dissolved inDMSO, it was then diluted serially with the incubation medium. The finalconcentration of DMSO in the wells was 1.2%. The sham-treated controlembryos were incubated in EM in the presence of 1.2% DMSO.

Microinjections were performed with the help of a SMZ-10A stereomicroscope (Nicon, Melville, N.Y.) and transjector 5246 (EppendorfScientific Inc, Westbury, N.Y. 11590) at RT. The eggs in chorions wererestrained in agar grooves filled with EM, and the drug containingsolution was introduced into the cytoplasm of one of the blastomeres ofthe two-cell stage eggs through a micropipette with a splinted sharp tipof 2-3 μm in diameter. All microinjections were performed under visualcontrol. Drugs for injection were dissolved in Hank'Balanced Salt Saline(Gibco, Rockville, Md.) with 10% DMSO. The volume of the solutioninjected into each blastomere was approximately 2 nl. For every drug,40-50 embryos were injected in 30 minutes of one series of experiment.After the treatment the embryos were transferred back to ST and studiedas above.

Observations of cell division and development of the ZF embryos werecarried out using a SMZ-10A stereo microscope (Nicon, Melville, N.Y.),once every 30 minutes within the first 3 hours of incubation and at 6,12 and 24 hours, as well. The drug effect was considered to be revealedwhen all embryos from one well were affected in a characteristic mannerin 3 independent experiments. The stereo microscope was fitted with aspecially designed transparent heating tray in order to keep embryos atST during observations. Pictures of the embryos were taken with a H-IIIPhotomicrographic System (Nicon, Melville, N.Y.) using Ektachrome 64Xfilm (Kodak, Rochester, N.Y.).

Cytotoxicity MTT Assays

The cytotoxicity of [VCp₂(acac)](CF₃SO₃) against human cancer cell lineswas performed as described previously (Ghosh et al, Clinc Canc Res,6:1536, 2000) using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay (Boehringer Mannheim Corp., Indianapolis,Ind.). Briefly, exponentially growing tumor cells were seeded into a96-well plate at a density of 2.5×10⁴ cells/well and incubated[VCp₂(acac)](CF₃SO₃) or 0.1% DMSO in PBS concentrations ranging from 0.1to 250 μM. Following incubation with drug for 48 hours at 37° C., toeach well, 10 μl of MTT (0.5 mg/ml final concentration) was added andthe plates were incubated at 37° C. for 4 hours to allow MTT to formformazan crystals by reacting with metabolically active cells. Theformazan crystals were solubilized overnight at 37° C. in a solutioncontaining 10% SDS in 0.01 M HCl. The absorbance of each well wasmeasured in a microplate reader (Labsystems) at 540 nm and a referencewavelength of 690 nm. The percent survival and the IC₅₀ values werecalculated using Graphpad Prism software version 2.0 (Graphpad Software,Inc., San Diego, Calif.), as described previously. Ghosh et al, ClincCanc Res, 6:1536, 2000.

Immunocytochemistry and Confocal Microscopic Analysis

At the appropriate time points coverslips containing BT-20 cells werefixed in −20° C. methanol for 15 minutes followed by 15 minutesincubation in phosphate buffered saline +0.1% Triton X-100 (PBS-Tx).Coverslips were next incubated with a primary antibody recognizingα-tubulin (Sigma, St. Louis Mo.) for 40 minutes in a humidified chamberat 37° C. Coverslips were washed for 15 minutes in PBS-Tx followed by a40 minutes incubation with a fluorescently labeled secondary antibody(Jackson Immunoresearch, West Grove, Pa.). The coverslips were againrinsed in PBS-Tx and incubated with 5 μM Toto-3 (Molecular Probes,Eugene Oreg.) for 20 minutes to label the DNA. Coverslips wereimmediately inverted onto slides in Vectashield (Vector Labs,Burlingame, N.H.) to prevent photobleaching, sealed with nail polish andstored at 4° C.

Slides were examined using a Bio-Rad MRC-1024 Laser Scanning ConfocalMicroscope mounted on a Nikon Eclipse E800 upright microscope with highnumerical aperture objectives. Slides were examined to determine thepercentage of cells in interphase vs. mitosis and representative imageswere taken. Digital data was processed using Lasersharp (Bio-Rad,Hercules, Calif.) and Photoshop (Adobe Systems, Mountain View, Calif.)software and printed on a Pictrography printer (Fuji Photo Elmsford,N.Y.)

Results

Embryonic development of the ZF (Danio rerio) is thoroughly studied andstaged (Westerfield, The Zebrafish Book, 1993, 2d Edition (Univ ofOregon Press, Eugene); Benyumov et al, Rus. J. Dev. Biol., 26(2):132,1995; Kimmel et al, Dev. Dynam., 203:253, 1995). In a meroblastic egg,cell divisions are rapid and occur after the ooplasmic segregation onthe animal pole of the egg cell, resulting within the first 3 hours ofdevelopment in generation of a multicellular blastula comprised ofseveral thousands of cells. The first series of cell divisions at theinitial cleavage stage are approximately synchronous, only 15 minutesapart, and each set of the-dividing blastomeres is characterized by adistinct pattern of cellular localization. This remarkable proliferationrate of undifferentiated eukaryotic vertebrate cells makes the ZF embryoan attractive experimental model for evaluation of new agents foranti-proliferative activity. In order to determine if the vanadiumcompounds such as [VCp₂(acac)](CF₃SO₃) could affect cell division, theeffect on embryonic development of Zebra fish was examined.

As discussed above, the embryos were dechorionated with Trypsin-EDTA andincubated in embryonic medium containing either vehicle (1.2% DMSO)alone or vehicle with [VCp₂(acac)](CF₃SO₃) at different concentrations.At standard temperature of 28.5° C., two-cell stage control embryosexposed to the vehicle alone reached 4-cell, 8-cell, and 64-cell stageswithin 15 minutes, 30 minutes, and 75 minutes, respectively. Within 3.5hours post fertilization, these embryos developed into a high blastulaand underwent gastrulation approximately 2.5 hours later.

[VCp₂(acac)](CF₃SO₃) inhibited cell division in aconcentration-dependent fashion. At 0.6 mM, it caused cell divisionblock at the 8-16 cell stage of embryonic development followed by totalcell fusion and developmental arrest. At lower concentrations, cellproliferation continued but was abnormal. Specifically, within 120minutes of incubation, cell division in the treated embryos was observedonly on top of the blastodisc and whereas the cells on periphery andcells adjacent to the yolk were totally fused. The treated eggs did notform a compact blastoderm and developed no further.

The anti-proliferative effects of [VCp₂(acac)](CF₃SO₃) microinjectedinto the cytoplasm of two-cell stage ZF eggs was next examined. At thelowest dose of 1 pmol/embryo, [VCp₂(acac)](CF₃SO₃) resulted in theformation of a blastocoel-like cavity in the blastoderm of lateblastulae and early gastrulae and disrupted further gastrulation. At adose of 40 pmols/cell, VDacac slowed down cell division and resulted indeterioration of the cell localization pattern and developmental block.Thus, vanadium compounds such as [VCp₂(acac)](CF₃SO₃) are a potentanti-mitotic agents.

The anti-mitotic effects of [VCp₂(acac)](CF₃SO₃) on proliferation ofhuman cancer cell lines was examined using MTT assays.[VCp₂(acac)](CF₃SO₃) inhibited the proliferation of the breast cancercell lines MDA-MB-231 and BT-20 as well as the glioblastoma cell lineU373 in a concentration-dependent fashion with IC₅₀ values of 9.6 μM,25.1 μM, and 35.7 μM, respectively.

The ability of [VCp₂(acac)](CF₃SO₃) to inhibit the proliferation ofhuman cancer cells prompted the hypothesis that this compound likelyaffects mitotic spindle formation. To test this hypothesis, we examinedthe mitotic spindles of vehicle-treated and [VCp₂(acac)](CF₃SO₃)-treatedBT-20 breast cancer cells using confocal laser scanning microscopy.Whereas in vehicle-treated control cells mitotic spindles were organizedas a bipolar microtubule array and the DNA was organized on a metaphaseplate, [VCp₂(acac)](CF₃SO₃)-treated BT-20 cells had aberrant monopolarmitotic structures where microtubules were detected only on one side ofthe chromosomes and the chromosomes were arranged in a circular pattern.These results provide a cogent explanation for the anti-miotic activityof [VCp₂(acac)](CF₃SO₃) against human cancer cells.

The data indicates that [VCp₂(acac)](CF₃SO₃) is a potentanti-proliferative agent, having disruptive effects on cell divisionduring mitosis.

The above specification, examples and data provide a completedescription of the manufacture and use of the composition of theinvention. Since many embodiments of the invention can be made withoutdeparting from the spirit and scope of the invention, the inventionresides in the claims hereinafter appended.

The above specification contains numerous references to literature andpatent publications, each of which is hereby incorporated by referenceas if fully set forth.

1. A method for inhibiting angiogenesis in a non-cancerous tissuecomprising administering to a subject an effective angiogenesisinhibiting amount of a vanadium compound having the following structure:

wherein, R₁ and R₂ are each independently a monodentate ligand ortogether form a bidentate ligand; R₃ and R₄ are each independently amonodentate ligand or together form a bidentate ligand; and R₅ is amonodentate ligand, or is absent.
 2. The method of claim 1, wherein eachmonodentate ligand is selected from the group consisting of halo, OH₂,O₃SCF₃, N₃, CN, OCN, SCN, SeCN, and a cyclopentadienyl ring, wherein thecyclopentadienyl ring is optionally substituted with one or more(C₁-C₃)alkyl, and each bidentate ligand is selected from the groupconsisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, Phen, or a derivativethereof.
 3. The method of claim 2, wherein each bidentate ligand isoptionally substituted with one or more of halo, (C₁-C₃) alkyl, (C₁-C₃)alkoxy, halo (C₁-C₃) alkyl, or a derivative thereof. 4.-5. (canceled) 4.The method of claim 2, wherein R₁ and R₂ are each independently halo. 5.The method of claim 4, wherein halo is chloro, bromo, or iodo.
 6. Themethod of claim 4, wherein halo is chloro. 9.-10. (canceled)
 7. Themethod of claim 1, wherein the vanadium compound has the followingstructure:

wherein R₁ and R₂ are each independently a monodentate ligand ortogether form a bidentate ligand; R₃ and R₄ together form a bidentateligand; and R₅ is a monodentate ligand, or is absent.
 8. The method ofclaim 7, wherein R₁ and R₂ are each independently a monodentate ligandselected from the group consisting of halo, OH₂, O₃SCF₃, N₃, CN, OCN,SCN, SeCN, and a cyclopentadienyl ring, wherein each cyclopentadienylring is optionally substituted with one or more (C₁-C₃)alkyl.
 9. Themethod of claim 8, wherein, R₃ and R₄ together form a bidentate ligandselected from the group consisting of acac, Bpy, Hfacac, Cat, Dtc, PH,H, Phen, and derrivatives thereof.
 10. The method of claim 7, wherein R₁and R₂ together form a bidentate ligand selected from the groupconsisting of acac, Bpy, Hfacac, Cat, Dtc, PH, H, Phen, and derrivativesthereof.
 15. (canceled)
 11. The method of claim 1, wherein said vanadiumcompound is: [VO(phen)], [VO(phen)₂], [VO(Me₂-phen)], [VO(Me₂-phen)₂],[VO(Cl-phen)], [VO(Cl-phen)₂], [VO(bipy)], [VO(bipy)₂], [VO(Me₂-bipy)],[VO(Me₂-bipy)₂], and [VO(Br,OH-acph)₂].
 12. A method for treatingdiabetic retinopathy in a subject, comprising administering to thesubject an effective mitosis inhibiting amount of a vanadium compoundhaving the following structure:

wherein, R₁ and R₂ are each independently a monodentate ligand ortogether form a bidentate ligand; R₃ and R₄ are each independently amonodentate ligand or together form a bidentate ligand; and R₅ is amonodentate ligand, or is absent.
 13. A method for treating restenosisfollowing coronary angioplasty in a subject, comprising administering tothe subject an effective amount of a vanadium compound having thefollowing structure:

wherein, R₁ and R₂ are each independently a monodentate ligand ortogether form a bidentate ligand; R₃ and R₄ are each independently amonodentate ligand or together form a bidentate ligand; and R₅ is amonodentate ligand, or is absent.
 14. A method for preventing ortreating diabetic retinopathy in a subject, comprising: administering tothe subject an effective mitosis inhibiting amount of a vanadiumcompound having the following structure:

wherein, R₁ and R₂ are each independently a monodentate ligand ortogether form a bidentate ligand; R₃ and R₄ are each independently amonodentate ligand or together form a bidentate ligand; and R₅ is amonodentate ligand, or is absent.
 14. The method of claim 14, whereinthe vascular injury is associated with an angioplasty procedure.
 15. Themethod of claim 14, wherein the compound is administered locally throughan implantable device.
 16. The method of claim 14, wherein saidadministering comprises administering the vanadium compound prior toinduction of vascular injury.
 17. The method of claim 14, wherein thecompound is administered before and after induction of vascular injury.18. The method of claim 14, wherein the vanadium compound isadministered at least two days before induction of vascular injury. 19.The method of claim 1 wherein the non-cancerous tissue is a vasculartissue.
 20. The method of claim 11 wherein the vascular tissue is acoronary artery.
 21. The method of claim 1 wherein the non-canceroustissue is a retina.
 22. The method of claim 1 wherein the non-canceroustissue is a tumor
 23. The method of claim 22 wherein the tumor is ahemangioma.
 24. The method of claim 19, wherein the angiogenesis isassociated with injury to the vascular tissue.
 25. The method of claim24, wherein the angiogenesis is associated with restenosis followinginjury to the vascular tissue.
 26. The method of claim 19, wherein thevascular tissue is a vessel.
 27. The method of claim 26, wherein thevessel is a coronary artery.
 28. The method of claim 26 wherein theinjury to the vessel is associated with balloon angioplasty, vesselstent, rotational and directional atherectomy, or laser angioplasty. 29.The method of claim 21, wherein the angiogenesis is associated withretinopathy.
 30. The method of claim 29, wherein the retinopathy isassociated with diabetes.